The invention relates to the laminin 12, laminin subunit xcex33, and laminin subunit xcex21, and methods of making and using these molecules.
The present invention is based, in part, on the discovery of a novel member of the laminin family, laminin 12. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of laminin 12 which includes an xcex12 subunit, a xcex21 subunit and a xcex33 subunit.
In a preferred embodiment, the xcex12 subunit has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human xcex12 subunit, e.g., the human xcex12 subunit of SEQ ID NO:7. The xcex12 subunit can be identical to a human xcex12 sequence, e.g., that of SEQ ID NO:7. In another embodiment, the xcex12 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:8. In addition, the xcex12 subunit can have substantially the same electrophoretic mobility as human xcex12 subunit, e.g., it appears as a 205 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an xcex12 subunit which is reactive with an xcex12-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 5H2. xcex12 specific antibodies can be made by methods known in the art.
Another preferred embodiment of the invention features a xcex21 subunit having at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human xcex21 subunit, e.g., the human xcex21 subunit of SEQ ID NO:9. Preferably, the xcex21 subunit has the identical amino acid sequence of human xcex21 subunit, e.g., that of SEQ ID NO:9. In another embodiment, the xcex21 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:10. In addition, the xcex21 subunit can have substantially the same electrophoretic mobility as human xcex21 subunit, e.g., it appears as a 185 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an xcex21 subunit which is reactive with an xcex21-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 545. xcex21-specific antibodies can be made by methods known in the art.
In yet another preferred embodiment, the xcex33 subunit of laminin 12 has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human xcex33 subunit, e.g., the xcex33 subunit of SEQ ID NO:3. The xcex33 subunit can be identical to a naturally occurring human xcex33 subunit, e.g., that of SEQ ID NO:3. In another embodiment, the xcex33 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4. In addition, the xcex33 subunit can have substantially the same electrophoretic mobility as human xcex33 subunit, e.g., it appears as a 170 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an xcex33 subunit which is reactive with an xcex33-specific antibody. xcex33-specific antibodies can be made by methods known in the art and taught herein.
In a preferred embodiment, the laminin 12 is a trimer which can be found in, or can be isolated from human placental chorionic villi. In another embodiment, the laminin 12 is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of laminin 12 in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode subunits of the laminin 12 trimer are expressed in a preferred cell-type by a tissue specific promoter, e.g., a milk specific promoter.
The present invention is also based, in part, on the discovery of a novel laminin subunit, xcex33. Accordingly, the invention features a recombinant or substantially pure or isolated preparation of a xcex33 polypeptide.
In a preferred embodiment, the xcex33 polypeptide has the following biological acitivities: 1) it promotes adhesion between tissue elements; 2) provides a site for insertion of nerves into the basement membrane. In other preferred embodiments: the xcex33 polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:3; the xcex33 polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:3; the xcex33 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the xcex33 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:3; the xcex33 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring xcex33 subunit; the xcex33 polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, xcex33 polypeptide.
In a preferred embodiment, the invention includes a xcex33 polypeptide encoded by a DNA insert of a plasmid deposited with ATCC as Accession No: 209357. In another embodiment, the xcex33 polypeptide is a polypeptide encoded by nucleotide sequences of the overlapping DNA inserts of more than one, preferably all seven of the plasmids deposited with ATCC as Accession No:209357.
In preferred embodiments: the xcex33 polypeptide is encoded by the nucleic acid in SEQ ID NO:4, or by a nucleic acid having at least about 85%, more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with the nucleic acid from SEQ ID NO:4.
In preferred embodiments, the xcex33 polypeptide includes a nidogen-binding domain. Generally, the nidogen-binding domain is at least 5 residues in length and preferably, has about 70, 80, 90, or 95% sequence identity with the nidogen-binding domain of the protein shown in SEQ ID NO: 3 (amino acid residues 750-755). In another embodiment, the xcex33 polypeptide includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins found in native xcex33 protein. In yet another embodiment of the invention features a xcex33 polypeptide that does not include or has an inactivated nidogen-binding domain which serves as an antagonist to xcex33 biological activities. Furthermore, a xcex33 polypeptide which has antagonist activity can have inactivated or excluded regions which comprise at least one cystein found in native xcex33 protein.
In a preferred embodiment, the xcex33 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 3. In other preferred embodiments, the xcex33 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO: 3. Preferably, the differences are such that: the xcex33 polypeptide exhibits a xcex33 biological activity, e.g., the 73 polypeptide retains a biological activity of a naturally occurring xcex33 subunit.
In preferred embodiments the xcex33 polypeptide includes a xcex33 subunit sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the xcex33 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 3, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO: 3.
In yet other preferred embodiments, the xcex33 polypeptide is a recombinant fusion protein having a first xcex33 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to xcex33. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment the xcex33 polypeptide includes amino acid residues 750-755 of SEQ ID NO:3. In another embodiment, the xcex33 polypeptide encodes domains IV-VI of the xcex33 subunit.
In preferred embodiments the xcex33 polypeptide has antagonistic activity, and is capable of: inhibiting adhesion between connective tissues.
In a preferred embodiment, the xcex33 polypeptide is a fragment of a naturally occurring xcex33 which inhibits connective tissue adhesion.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The xcex33 polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed xcex33 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a xcex33 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the xcex33 polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 3.
The present invention also includes an antibody preparation specifically reactive with an epitope of the xcex33 immunogen or generally of a xcex33 polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:3, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
In preferred embodiments the xcex33-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of 170 kDa as determined by SDS-PAGE.
In another embodiment, the xcex33 polypeptide comprises amino acid residues 100-1761 of SEQ ID NO: 3.
In a preferred embodiment, the xcex33 polypeptide has one or more of the following characteristics:
(i) it has the ability to promote adhesion between connective tissues;
(ii) it has a niolecular weight, amino acid composition or other physical characteristic of xcex33 subunit of SEQ ID NO:3;
(iii) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a xcex33 polypeptide of SEQ ID NO:3;
(iv) it can be isolated from human placenta chorionic villi;
(v) it has a nidogen-binding domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues 750-755 of SEQ ID NO:3;
(vi) it can colocalize with protein ubiquitin carboxy terminal hydroxylase I;
(vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found amino acid sequence of native xcex33.
Also included in the invention is a composition which includes a xcex33 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a xcex33 polypeptide, e.g., a xcex33 polypeptide described herein.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:4. In other preferred embodiments, the xcex33 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:4. In another preferred embodiment, the xcex33 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:4.
In preferred embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of at least one and preferably all of the DNA inserts of the plasmids deposited with ATCC as Accession No: 209357.
In preferred embodiments, the subject xcex33 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the xcex33 gene sequence (also referred to as LAMG3), e.g., to render the xcex33 gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a xcex33 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4. More preferably, the nucleic acid probe corresponds to at least 20 consecutive nucleotides from SEQ ID NO: 4.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 4, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a xcex33 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a xcex33 subunit transgene, or which otherwise misexpress a xcex33 gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a xcex33 transgene, e.g., a heterologous form of a xcex33 gene, e.g., a gene derived from humans (in the case of a non-human cell). The xcex33 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous xcex33 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed xcex33 alleles or for use in drug screening.
In another aspect, the invention features a transgenic xcex33 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a xcex33 gene, e.g., a gene derived from humans. In a further embodiment, the xcex33 transgene includes a tissue specific promoter, e.g., a milk-specific promoter. In other preferred embodiments, the animal has an endogenous xcex33 gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed xcex33 alleles or for use in drug screening.
The invention is also based, in part, on the discovery of a novel laminin subunit, xcex24. Accordingly, the invention features a recombinant or substantially pure preparation of a xcex24 polypeptide.
In preferred embodiment, the xcex24 polypeptide has the following biological activities: 1) it promotes adhesion between tissue elements; 2) it aids in wound healing. In other preferred embodiments: the xcex24 polypeptide includes an amino acid sequence with at least 65%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO: 1; the xcex24 polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO: 1; the xcex24 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the xcex24 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO: 1; the xcex24 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring xcex24 subunit; the xcex24 polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, xcex24 polypeptide.
In preferred embodiments: the xcex24 polypeptide is encoded by the nucleic acid in SEQ ID NO:2, or by a nucleic acid having at least about 65% to about 70%, more preferably at least 80%, even more preferably at least about 90% to about 95%, and most preferably about 99% sequence identity with the nucleic acid from SEQ ID NO: 2.
In preferred embodiments, the xcex24 polypeptide includes domains VI and V found in native xcex24 subunits. Amino acid residues from about 221-262 and 263-535 of SEQ ID NO: 1 are exemplary of domains VI and V, respectively, of xcex24. Generally, domain VI is at least 33 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%, and most preferably about 90% to about 95% sequence identity with the amino acid residues 221-262 of the xcex24 protein shown in SEQ ID NO: 1. Domain V is at least 272 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%, and most preferably about 90% to about 95% sequence identity with the amino acid residues 263-535 of the xcex24 protein shown in SEQ ID NO: 1. In another embodiment, the xcex24 polypeptide has at least 5, preferably 6 or 7, and most preferably 8 cysteins as found in native xcex24. In yet another embodiment, a xcex24 polypeptide which has antagonist activity has inactivated or excluded regions which comprise at least one of the cysteins found in native xcex24 protein.
In a preferred embodiment, the xcex24 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 1. In other preferred embodiments, the xcex24 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO: 1. Preferably, the differences are such that: the xcex24 polypeptide exhibits a xcex24 biological activity, e.g., the xcex24 polypeptide retains a biological activity of a naturally occurring xcex24 subunit.
In preferred embodiments the xcex24 polypeptide includes a xcex24 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the xcex24 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 1, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:1.
In yet other preferred embodiments, the xcex24 polypeptide is a recombinant fusion protein having a first xcex24 portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to xcex24. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In preferred embodiments the xcex24 polypeptide has antagonistic activity, and is capable of: inhibiting the adhesion of connective tissues.
Preferably, the xcex24 polypeptide is a fragment of a naturally occurring xcex24 which inhibits connective tissue adhesion.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. In one aspect of the invention, the xcex24 polypeptide is a splice variant of the xcex24 subunit. In another preferred embodiment, the xcex24 splice variant is encoded by a nucleic acid molecule identical to the nucleotide sequence of SEQ ID NO:6. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed xcex24 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a xcex24 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the xcex24 polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO: 1.
The present invention also includes an antibody preparation specifically reactive with an epitope of the xcex24 immunogen or generally of a xcex24 polypeptide, preferably an epitope which consists all or in part of residues from the amino acid sequence of SEQ ID NO: 1, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
In preferred embodiments the xcex24-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has an estimated molecular weight of 200 kDa as determined by SDS-PAGE.
In a preferred embodiment, the xcex24 polypeptide has one or more of the following characteristics:
(i) it has the ability to promote adhesion between connective tissues;
(ii) it has a molecular weight, amino acid composition or other physical characteristic of xcex24 subunit of SEQ ID NO: 1;
(iii) it has an overall sequence similarity of at least 50%, preferably at least 65%, more preferably at least 70, 80, 90, or 95%, with a xcex24 polypeptide of SEQ ID NO: 1;
(iv) it can be isolated from human placenta chorionic villi;
(v) it can associate with xcex13 or xcex32 subunits;
(vi) it has coiled coils in domains I and II.
(vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found in native xcex24 sequence.
Also included in the invention is a composition which includes a xcex24 polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition for in vitro and in vivo pharmaceutical or veterinary use. Such uses can include aiding in wound healing or promotion of the adhesion of dermal and epidermal cells.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a xcex24 polypeptide, e.g., a xcex24 polypeptide described herein.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 65% sequence identity to a nucleotide sequence of SEQ ID NO:2. In other preferred embodiments, the xcex24 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least 70%, preferably 80%, more preferably about 90% to about 95%, and even more preferably about 99% sequence identity to the nucleotide sequence from SEQ ID NO:2. In another preferred embodiment, the xcex24 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:2.
In preferred embodiments, the subject xcex24 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer *sequence, operably linked to the xcex24 gene sequence (also referred to as LAMB4), e.g., to render the xcex24 gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a xcex24 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:2, more preferably to at least 20 consecutive nucleotides from SEQ ID NO:2.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of SEQ ID NO:2, nucleotides 4686-5870.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 2, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a xcex24 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a xcex24 transgene, or which otherwise misexpress a xcex24 gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a xcex24 transgene, e.g., a heterologous form of a xcex24 gene, e.g., a gene derived from humans (in the case of a non-human cell). The xcex24 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous xcex24 gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed xcex24 alleles or for use in drug screening.
In another aspect, the invention features a transgenic xcex24 animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a xcex24 gene, e.g., a gene derived from humans. In a further embodiment, the xcex24 transgene includes a tissue specific promoter, e.g., a milk-specific promoter. In other preferred embodiments, the animal has an endogenous xcex24 gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed xcex24 alleles or for use in drug screening.
In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., laminin 12, or xcex33 (or a laminin trimer which includes xcex33), sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.
In preferred embodiments the first tissue is healthy tissue, e.g., skin tissue, and the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, e.g., burned or abraided tissue.
In preferred embodiments the first tissue and second tissue element are normally adhered but have become detached from one another due to trauma, burn or other physical injury, disease, or age.
In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve; the first tissue element is a retinal cell or retina tissue and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the a retinal cell or retina tissue, the first tissue is a nerve and the second tissue is basement membrane.
The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, xcex33 (or a laminin trimer which includes xcex33), sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
The wound can be in any tissue, but preferably ina tissue in which the laminin normally occurs. Examples skin, central or peripheral nervous tissue, tissues of the eye, e.g., the retinal, the basement membrane, or any tissue which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered thereto.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraided.
The administration of laminin can be repeated.
In another aspect, the invention feature""s a method of promoting nerve growth or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., laminin 12, or xcex33 (or a laminin trimer which includes xcex33), sufficient to promote nerve growth or regeneration. The administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the nerve growth or regeneration is promoted at a wound site.
The administration of laminin can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a laminin described herein, e.g., xcex33 or laminin 12.
Such disorders include, e.g., a disorder associated with the misexpression of a laminin e.g., laminin 12, or misexpression of the xcex33 subunit; a disorder of the central or peripheral nervous system; a disorder associated with a genetic lesion at chromosome 9, region q31-34; Fukuyama-type muscular dystrophy; muscle-eye-brain disease; Walker-Warburg Syndrome (hydrocephalus, ageria, and retinal displasia); a retinal disorder, e.g, retinitis pigmentosa-deafness syndrome (which may be a subtype of Walker-Warburg Syndrome); a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, e.g., an epidermal or dermal, disorder; a disorder associated with the testis, spleen, placenta, thymus, ovary, small intestine, lung, or liver.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the xcex33 gene, or other gene which encodes a subunit of laminin 12, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the xcex33 gene, or other gene which encodes a subunit of laminin 12;
detecting, in a tissue of the subject, the misexpression of the xcex33 gene, or other gene which encodes a subunit of laminin 12 at the mRNA level, e.g., detecting a non-wild type level of a xcex33, or an other laminin 12 subunit mRNA; detecting, in a tissue of the subject, the misexpression of the xcex33 gene, or other gene which encodes a subunit of laminin 12, at the protein level, e.g., detecting a non-wild type level of a xcex33, or an other laminin 12 subunit polypeptide.
In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the xcex33 gene, or other gene which encodes a subunit of laminin 12; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the LAMG3 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the xcex33 gene, or other gene which encodes a subunit of laminin 12; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the xcex33 gene, or other gene which encodes a subunit of laminin 12; or a non-wild type level of xcex33, or other subunit of laminin 12.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments the method includes determining the structure of a xcex33 gene, or other gene which encodes a subunit of laminin 12, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample form the subject with an antibody to the laminin protein or a nucleic acid which hybridizes specifically with the xcex33 gene, or other gene which encodes a subunit of laminin 12.
In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, e.g., xcex24, sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements include, skin cells, e.g., epidermal or dermal cells, neuronal cells, e.g., nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.
In preferred embodiments the first tissue is healthy tissue, e.g., skin tissue, and the second tissue is wounded, e.g., burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, e.g., burned or abraided tissue.
In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve; the first tissue is a nerve and the second tissue is basement membrane.
The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., xcex24, sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
The wound can be in any tissue, but preferably in a tissue in which the laminin normally occurs in fetal or adult life. Examples examples include skin the basement membrane.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraded.
The administration of laminin can be repeated.
In another aspect, the invention features a method of promoting tissue growth, development, or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, e.g., xcex24, sufficient to promote tissue growth, development, or regeneration in a subject. The administration can be directed to the site where nerve growth or regeneration is desired, e.g., by topical appication or by injection, or administered in a systemic fashion.
In preferred embodiments the molecule is exogenous (e.g., administered to a subject) or is recombinant.
In preferred embodiments the nerve growth or regeneration is promoted at a wound site.
The administration of laminin can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a laminin molecule described herein, e.g., xcex24.
Such disorders include, e.g., a disorder associated with the misexpression of a laminin, e.g., xcex24; a disorder associated with a genetic lesion at chromosome region 7q22-q31.2; a developmetnal disorder; a disorder associated with abnormal levels, e.g., abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, e.g., an epidermal or dermal, disorder.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the (4 gene, e.g, detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the xcex24 gene;
detecting, in a tissue of the subject, the misexpression of the xcex24 gene, e.g., detecting a non-wild type level of a xcex24 mRNA;
detecting, in a tissue of the subject, the misexpression of the xcex24, at the protein level, e.g., detecting a non-wild type level of a xcex24 polypeptide.
In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the xcex24; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the xcex24 gene, a gross chromosomal rearrangement of the xcex24 gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:2, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the LAMB4 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments: detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the xcex24; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the xcex24; or a non-wild type level of xcex24.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments the method includes determining the structure of the xcex24, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample form the subject with an antibody to the xcex24 protein or a nucleic acid which hybridizes specifically with the xcex24.
In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, e.g., bind, a subject laminin polypeptide, e.g., laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide. This method can be performed in vitro, e.g., in a cell free system or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject laminin polypeptide. It can also be used to find natural or synthetic inhibitors of subject laminin polypeptide.
In another aspect, the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occurring substrate to which binds a subject laminin polypeptide, e.g., of laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24, for the ability to bind a subject laminin polypeptide. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin polypeptide, e.g., the ability of the compound to inhibit a subject laminin polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
In another aspect, the invention features, a method of evaluating a first compound, e.g., a subject laminin polypeptide, e.g., laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24, for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or substrate to which binds a subject laminin polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.
In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject laminin polypeptide, e.g., of laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand which binds a subject laminin polypeptide, or a fragment thereof. The method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject laminin polypeptide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject laminin polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject laminin polypeptide. A change, e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject laminin polypeptide. In preferred embodiments: the second polypeptide, and the subject laminin polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject laminin polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay).
In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject laminin polypeptide subject laminin polypeptide, e.g., of laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or a substrate to which binds a subject laminin polypeptide, or a fragment thereof. The method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject laminin polypeptide, with a second polypeptide.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject laminin polypeptide, e.g., a laminin 12, xcex33, a laminin trimer which includes xcex33, p4, or a laminin trimer which includes xcex24 polypeptide regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making a xcex33 or xcex24 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring xcex33 or xcex24 polypeptide, e.g., a naturally occurring xcex33 or xcex24 polypeptide. The method includes: altering the sequence of a xcex33 or xcex24 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of a xcex33 or xcex24 polypeptide having a biological activity of a naturally occurring xcex33 or xcex24 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a xcex33 or ,B4 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject laminin polypeptide, e.g., a laminin 12, xcex33, a laminin trimer which includes xcex33, xcex24, or a laminin trimer which includes xcex24.
In another aspect, the invention includes: a xcex33, xcex24 nucleic acid, e.g., a xcex33, xcex24 nucleic acid inserted into a vector; a cell transformed with a xcex33, xcex24 nucleic acid; a xcex33, xcex24 made by culturing a cell transformed with a xcex33, xcex24 nucleic acid; and a method of making a xcex33, xcex24 polypeptide including culturing a a cell transformed with a xcex33, xcex24 nucleic acid.
The inventors have shown that xcex33 forms laminin 12 in association with xcex12 and xcex21. However, we are unsure of the chain associations of xcex33 within other tissues. It is very likely that xcex33 can also associate with xcex33, xcex13, xcex14, and xcex15; with xcex22, xcex23, xcex24 and xcex25. Therefore, our results predict 25 new laminins: laminins 12-37. xcex33 and xcex24 polypetides of the invention can be expressed with, assembled with, or administered with other laminin subunits in any of the methods described herein. E.g., xcex33 can be assembled with an xcex1 and a xcex2 subunit to form a laminin trimer. xcex24 can be assembled with an xcex1 and a xcex2 subunit to form a laminin trimer.
In any treatment or therapeutic application which administers xcex33, a xcex22 subunit can also be administered.
A xe2x80x9cheterologous promoterxe2x80x9d, as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d or isolated xe2x80x9cpreparationxe2x80x9d of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 xcexcg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpure nucleic acidxe2x80x9d, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
xe2x80x9cSequence identity or homologyxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions comparedxc3x97100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more subject laminin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
xe2x80x9cUnrelated to a xcex33 or xcex24 amino acid or nucleic acid sequencexe2x80x9d means having less than 30% sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occurring xcex33 or xcex24 sequence disclosed herein.
A polypeptide has xcex33 biological activity if it has one or more of the properties of xcex33 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the properties of xcex33 disclosed herein.
A polypeptide has xcex24 biological activity if it has one or more of the properties of xcex24 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the properties of 4 disclosed herein.
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
Subject, as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.
As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding a xcex33 or 4 polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, Such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embtyo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.